Project Circuit Design
Overview
This year, our project mainly concerns about the crucial pathogenic organism nematode. We intend to build a kind of novel engineering bacteria which will express organics to allure nematodes, and synthesis specific toxin to kill the pests after they devour them. Hereby we are able to build an environmentally friendly system of killing the nematodes.
Although bace16 and rMpL are the specific toxin of killing the nematodes, if expressed too much, they will undeniably do harm to the ecosystem of the soil itself. What’s more, taking the economic efficiency into consideration, if releasing the toxin continuously, we will not receive the maximum economic benefits. Therefore, we construct a circuit controlled by light to regulate the releasing of the toxin. The loop consists of three parts: 1. Photoinduction System; 2. Reverse System；3. Trap and Kill
In this circuit, we achieve the following effects. When bacteria are under the light, the promoter OmpC is inhibited. Meanwhile the promoter Pcon expresses limonene constantly to allure nematodes. However, when the amount of the nematodes around is large enough, the “light ” will be turn off. At the same time, the promoter OmpR starts the expression of the int intgrase to make the promoter Pcon reverse. So the expression of the limonene is turned off while the expression of the toxin is started. The advantages of this loop is shown as follows:
- Steerable: we can control the expression of the inducers and the toxin by regulating the light.
- Environmentally friendly: Only when the nematodes gathering around reaches a considerable number, the expression of the toxin will start. Therefore, the concentration of the toxin can be limited at a reasonable range.
- Economize: The expression of the toxin as well as the release of the bait are two different stages during the trapping and killing. So we could use less E.coli to kill the nematodes.
- Avoiding of Overload: By regulating the two pathway of toxin and Bait, we have reduced the load of metabolism for E.coli.
Photoinduced System
cph8
The red light sensor (Cph8) is a fusion protein which consists of a phytochrome Cph1 and a histidine kinase domain, Envz-OmpR. Cph1 is the first member of the plant photoreceptor family that has been identified in bacteria. EnvZ-OmpR, a dimeric osmosensor, is a multidomain transmembrane protein. Cph1 can be inactivated under red light. Upon changes of extracellular osmolarity, EnvZ specifically phosphorylates its cognate response regulator OmpR, which, in turn, regulates the PompC. Cph8 can serve as a photoreceptor that regulates gene expression through PompC. Without red light, Cph1 is activated and it enables EnvZ-OmpR to autophosphorylate which in turn activates PompC. Under the exposure of red light, however, Cph1 is inactivated, inhibiting the autophosphorylation, thus turning off gene expression.
We insert RBS(B0035) and constitutive promoter(J23100) in front of the Cph8 sequences(K592000) to make sure that Cph8 can be expressed inside the E.coli.
promoter OmpC
PompC is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC gene. Phosphorylated OmpR binds to the three operator sites and activates transcription.
We insert PompC(BBa_R0082) sequences into the upstream region of our target gene gp35 through cleavage and ligation, which together is connected to vector PSB1C3 afterwards, and therefore we are able to regulate the expression of gp35 through the control of light.
pcyA+ho1
Moreover, in order to function well, Cph8 has to form chromophore with PCB biosynthetic genes (BBa_I15008 and BBa_I15009), where the formation of PCB requires the gene pcyA and ho1. Ho1 is a sort of heme oxygenase from Synechocystis which can oxidize the heme group using a ferredoxin cofactor, generating biliverdin IXalpha. And then, PcyA, a kind of ferredoxin oxidoreductase from Synechocystis, functions to turn biliverdin IXalpha (BV) into PCB.
We used overlap PCR to connect gene pcyA（BBa_I15009）and ho1（BBa_I15008）together along with the constitutive promoter(BBa_J23100) to ensure a continuous work of the photoinduced system in our E.coli.
Bidirectional Transcriptional System
Bxb1 gp35 is a serine integrase in Mycobacterium phage Bxb1. This intergrase can exclusively catalyze site-specific recombination. The dismiss of light singal can drive the expression of integrase, and results in the reverse of the sequence（there it means the promoter Pcon） between attB and attP, changing the two sites to attL and attR at the meantime. As a consequence, the promotor moves on to express the toxic protein instead of the attractant.
We insert the gp35 gene into the downstream region of the PompC promotor by means of 3A Assembly so that the expression of integrase can be regulated by light. In addition, we add a 6N promotor between PompC and gp35 to decrease basal integrase expression above flipping threshold level which could lower the translation efficiency of gp35. Meanwhile, we insert the recombined locus of attB and attP into the flanking sequences of our constitutive promotor (BBa_J23110 ) and so far we have accomplished our reverse promotor system.
Trap and Kill System
We add a bait gene downstream attP at the constitutive promoter as well as adding the gene of toxin to the attB. When it is under light, the expression of gp35 is inhibited. The promoter turn to the original direction, so it starts expressing the attractive organics. Nevertheless when the light disappears, the expression of gp35 begins with the help of PompC, the direction changes to express the toxin. So our project of building the light-induced system finished.